Recent evidence indicates that one of the important biochemical events in experimental myocardial infarction is the loss of pyridine nucleotides from the infarcted area. The loss of NAD ion, particularly from the mitochondria, would explain the decreased ability of the tissue to respire and engage in oxidative phosphorylation, the major metabolic sequence by which ATP is synthesized in the heart. Because most of the dehydrogenases responsible for the oxidation of citric acid cycle intermediates require NAD ion as an essential cofactor, ATP synthesis would remain impaired if NAD ion is depleted, even when normal oxygen tension is restored. While it is clear that maintenance of proper levels of pyridine nucleotides is critical to the cell, at present, virtually nothing is known about how this is accomplished. Moreover, the origin of mitochondrial NAD ion is also unknown because intact mitochondria are impermeable to pyridine nucleotides. In the research that is proposed, isolated heart mitochondria and other subcellular components will be examined for the presence of the enzymes that are involved in the biosynthesis of NAD ion. The assays will entail incubating the preparations with radioactive precursors and isolating the radioactive products by chromatographic methods. The biosynthetic enzymes will also be isolated and studied in purified form by kinetic methods to uncover properties that may be of regulatory significance. A knowledge of how pyridine nucleotides are regulated and how alterations in the regulatory mechanisms are related to ischemic disease may help identify possible points of therapeutic intervention that may be utilized to minimize the changes in NAD ion levels that accompany infarction.